This technique has overtaken the conventional slow freezing technique; and we use this routinely to cryopreserve (freeze) supernumerary eggs and embryos, because it has a much better survival rate.

Preserving eggs and embryos

Vitrification can efficiently preserve spare eggs and embryos, so they can be used later on to achieve a pregnancy after thawing. Survival rates after vitrification and subsequent pregnancy rates are much better than they were with conventional slow freezing.

This describes the laboratory protocol we use for freezing eggs and embryos.The principle is simple. Vitrification is dependent on the placement of the oocyte/embryo in a very small volume of vitrification medium which is then cooled at an extreme rate, so that the embryos are flash frozen and preserved intact.

MATERIALS REQUIRED

Media : CRYOTECH VITRIFICATION KIT

The CRYOTECH VITRIFICATION KIT is a TWO-step freezing system containing Cryobase buffer with ethylene glycol, DMSO and trehalose as the cryoprotectants.

The kit has 2 Solutions

• Vitrification Step 1(Equilibration Solution): 7.5% DMSO and 7.5% Ethylene glycol
• Vitrification Step 2(Vitrification Solution): 15% DMSO and 15% Ethylene glycol

Disposables :

1. BD Falcon 1006 Dishes
2. Flexipet 170 u
3. Cryolocks
4. Goblets
5. Thermocol Box
6. Liquid nitrogen (LN2)
7. Forceps for handling Cryolocks in LN2

Preparation

The Whole procedure is performed under room temperature (25-27 deg C).

FREEZING PROTOCOL

Equilibration of oocytes and Embryos

Vitrification

Attention :

The following steps must be made in no less than 25 sec and a maximum time of 90 sec.

Note : loading embryos onto cryolock is the most difficult part in the procedure. One has to make sure that the volume of media should be as low as possible, since the freezing medium a has high concentration of cryoprotectant and is embryotoxic, which can affect the survival of embryos.

1. Fill the vitri plate with 300ul of ES in the 1st well, and 300 ul of VS in the 2nd and 3rd Well.
2. Bring ES and VS to room temperature (at least 1 hour before vitrification)
3. Take culture dish containing oocyte/embryo out from the incubator.
4. Aspirate the oocyte/embryo at the middle of the fine part of the flexipet (140u for oocyte/embryo and 300u for blastocysts)
5. Put the oocytes /Embryos on the surface of the ES in the 1st Well. Start the stop watch.
6. The oocyte/embryo will sink and begin to shrink, and gradually returns to the original size (maximum 15 min. for oocytes and blastocysts and 12 min. for other stages of embryos
   • Aspirate the oocyte/embryo and ES until the middle of the flexipet.
   • Blow out oocyte/embryo at the middle depth of VS1 with ES (step1)
   • Expel remained ES inside the flexipet and aspirate fresh VS1 from the edge of the wall.
   • Oocyte/embryo floats immediately to the surface of VS1. Aspirate oocyts/embryo at the top inside of pipette.
   • Blow it again to the bottom of VS1.
   • The oocyte/embryo floats slowly to the middle depth and stop.
   • Expel remained VS1 inside the flexipet and aspirate fresh VS2. Aspirate oocyte/embryo at the top inside of the flexipet.
   • Blow the oocyte/embryo to the middle depth of VS2.
   • Expel remained VS inside the flexipet and aspirate fresh VS2 from the edge of the Solution.
   • Blow VS2 and mix the solution around oocyte/embryo to exchange the remaining previous solution.
   • Expel and wash inside of the flexipet with fresh VS2.
   • Place oocyte/embryo near the end of cryolock with minimal volume of VS2.
   • Immediately submerge the cryolock into fresh liquid nitrogen.
   • Put the cap on the cryolock in the liquid nitrogen.
   7. Put all the cryolocks into Goblet and place the goblet into the canister.

WARMING

Materials

Cryotech vitrification warming kit

• Warmins solution (TS)
• Diluent Solution (DS)
• Washing Solution (WS)
• 1 warm plate with 4 wells
• Flexipet 170u
• Forceps

Preparation

• Place the warm plate and TS vial in the incubatore at 37 deg C for more than 3 hours before warming (overnight storage is recommended)
• Bring DS and WS vials to room temperature (25-27 deg C) at least 1 hour before warming.
• Prepare fresh liquid nitrogen in a thermocol box.
• Take patients cryolock out from the cryocan.
• Take warm plate and TS out of the incubator and fill the rectangular well with TS.

Warming (1 min.)

• Quickly put the cryolock into TS well (within 1 sec.)
  Start the stop watch for 1 min.
• Oocyte / embryo separates from the cryotec sheet by itself and begin to float.
• Confirm the oocyte/embryo existende in TS Well.

Dilution (3 min.)

• Aspirate oocyte /embryo and 3mm long TS into the flexipet.
• Blow out TS to the bottom centre of DS and expel the oocyte/embryo slowly bottom of the TS
  TS layer in DS well.
  Start the Stop watch for 3 min.

Washing 1 (5min.)

• Aspirate the oocyte / embryo and 3 mm long of DS into the flexipet.
• Blow our DS into the Bottom center of WS1 and expel the oocyte/embryo slowly bottom of DS layer in WS1 Well.

Washing 2

• Aspirate the oocyte/embryo with minimal volume of WS1.
• Put the oocyte/embryo to surface of WS2.
• When oocyte/embryo sinks to the bottom of WS2 , aspirate and put on the surface to wash for 2 times.
• Put the oocyte / embryo in droplet of the culture medium for ICSI and ET.